1. Field of the Invention
This invention is in the field of biologically active compounds.
2. Discussion of the Background
The prior art is replete with examples of chemically, microbially, or enzymatically synthesizing compounds with biological activity. The goal of these efforts is the discovery of new and improved pharmaceutical compounds.
The discovery of new pharmaceutical compounds is for the most part a trial and error process. So many diverse factors constitute an effective pharmaceutical compound that it is extremely difficult to reduce the discovery process to a systematic approach. Typically, thousands of organic compounds must be isolated from biological sources or chemically synthesized and tested before a pharmaceutical compound is found.
Synthesizing and testing new compounds for biological activity, which is the first step in identifying a new synthetic drug, is a time consuming and expensive undertaking. Typically, compounds must by synthesized, purified, tested and quantitatively compared to other compounds in order to identify active compounds or identify compounds with optimal activity. The synthesis of new compounds is accomplished for the most part using standard chemical methods. Such methods provide for the synthesis of virtually any type of organic compound; however, because chemical reactions are non-specific, these syntheses require numerous steps and multiple purifications before a final compound is produced and ready for testing.
New biological and chemical approaches have recently been developed which provide for the synthesis and screening of large libraries of small peptides and oligonucleotides. These methods provide for the synthesis of a broad range of chemical compounds and provide the means to potentially identify biologically active compounds. The chemistries for synthesizing such large numbers of these natural and non-naturally occurring polymeric compounds is complicated, but manageable because each compound is synthesized with the same set of chemical protocols, the difference being the random order in which amino acids or nucleotides are introduced into the reaction sequence.
The prior art is replete with examples showing enzymatic conversion of non-physiological substances under many conditions.
References Demonstrating that Enzyme Specificity can be Changed/Tailored
1. Zaks, A. and Klibanov, A. M. Substrate specificity of enzymes in organic solvents vs. water is reversed. Journal of the American Chemical Society 108 2767-2768, 1986. PA1 2. Ferjancic, A., Puigserver, A. and Gaertner, H. Unusual specificity of PEG-modified thermolysin in peptide synthesis catalyzed in organic solvents. Biotechnology Letters 10 (2) 101-106, 1988. PA1 3. Nasri, M. and Thomas, D. Increase of the potentialities of restriction endonucleases by specificity relaxation in the presence of organic solvents. Ann. N.Y. Acad. Sci. 542 255-265, 1988. PA1 4. Stahl, M., Mansson, M. O. and Mosbach, K. The synthesis of a D-amino acid ester in an organic media with chymotrypsin modified by a bio-imprinting procedure. Biotechnology Letters 12 (3) 161-166, 1990. PA1 5. Stahl, M., Jeppsson-Wistrand, U., Mansson, M. O. and Mosbach, K. Induced stereoselectivity and substrate selectivity of bio-imprinted .alpha.-chymotrypsin in anhydrous organic media. Journal of the American Chemical Society 113 (24) 9366-9368, 1991. PA1 6. Gololobov, M. Y., Voyushina, T. L., Stepanov, V. M. and Adlercreutz, P. Organic solvent changes the chymotrypsin specificity with respect to nucleophiles. FEBS Letters 307 (3) 309-312, 1992. PA1 7. Hertmanni, P., Pourplanche, C. and Larreta-Garde, V. Orientation of enzyme catalysis and specificity by water-soluble additives. Ann. New York Acad. Sci. (Enzyme Eng. XI, D. S. Clark, D. A. Estell, eds) 672 329-335, 1992. PA1 8. Cabezas, M. J., del Campo, C., Llama, E., Sinisterra, J. V. and Gaertner, H.. Organic reactions catalyzed by modified enzymes. 1. Alteration of the substrate specificity of a-chymotrypsin by the modification process. Journal of Molecular Catalysis 71 (2) 261-278, 1992. PA1 9. Nagashima, T., Watanabe, A. and Kise, H. Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity. Enzyme and Microbial Technology 14 (10) 842-847, 1992. PA1 10. Parida, S. and Dordick, J. S. Tailoring lipase specificity by solvent and substrate chemistries, J. Org. Chem. 58 (12) 3238-3244, 1993. PA1 11. Tawaki, S. and Klibanov, A. M. Chemoselectivity of enzymes in anhydrous media is strongly solvent dependent. Biocatalysis 8 (1) 3-19, 1993. PA1 12. Wescott, C. F. and Klibanov, A. M. Solvent variation inverts substrate specificity of an enzyme. JACS 115 (5) 1629-1631, 1993. PA1 1. Sakurai, T., Margolin, A. L., Russell, A. J. and Klibanov, A. M. Control of enzyme enantioselectivity by the reaction medium. Journal of the American Chemical Society 110 (21) 7236-7237, 1988. PA1 2. Fitzpatrick, P. A. and Klibanov, A. M. How can the solvent affect enzyme enantioselectivity? Journal of the American Chemical Society 113 (8) 3166-3171, 1991. PA1 3. Hult, K. and Norin, T. Enantioselectivity of some lidases--control and prediction. Pure and Applied Chemistry 64 (8) 1129-1134, 1992. PA1 4. Miyazawa, T., Kurita, S., Ueji, S., Yamada, T. and Kuwata, S. Resolution of racemic carboxylic acids via the lipase-catalyzed irreversible transesterification using vinyl esters--effects of alcohols as nucleophiles and organic solvents on enantioselectivity. Biotechnology Letters 14 (10) 941-946, 1992. PA1 5. Tawaki, S. and Klibanov, A. M. Inversion of enzyme enantioselectivity mediated by the solvent. Journal of the American Chemical Society 114 (5) 1882-1884, 1992. PA1 6. Ueji, S., Fujino, R., Okubo, N., Miyazawa, T., Kurita, S., Kitadani, M. and Muromatsu, A. Solvent-induced inversion of enantioselectivity in lipase-catalyzed esterification of 2-phenoxypropionic acids. Biotechnology Letters 14 (3) 163-168, 1992. PA1 7. Terradas, F., Testonhenry, M., Fitzpatrick, P. A. and Klibanov, A. M. Marked dependence of enzyme prochiral selectivity on the solvent. Journal of the American Chemical Society 115 (2) 390-396, 1993. PA1 8. Herradon, B. Biocatalytic synthesis of chiral polyoxygenated compounds: effect of the solvent on the enantioselectivity of lipase catalyzed transesterifications in organic solvents. Synlett 2 108-110, 1993. PA1 1. Bianchi, D., Cesti, P., Golini, P., Spezia, S., Garavaglia, C. and Mirenna, L. Enzymatic preparation of optically active fungicide intermediates in aqueous and in organic media. Pure and Applied Chemistry 64 (8) 1073-1078, 1992. PA1 2. Natoli, M., Nicolosi, G. and Piattelli, M. Regioselective alcoholysis of flavonoid acetates with lipase in an organic solvent. Journal of Organic Chemistry 57 (21) 5776-5778, 1992. PA1 3. Izumi, T., Tamura, F. and Sasaki, K. Enzymatic kinetic resolution of &lt;4&gt;(1,2)ferrocenophane derivatives. Bulletin of the Chemical Society of Japan 65 (10) 2784-2788, 1992. PA1 4. Miyazawa, T., Mio, M., Watanabe, Y., Yamada, T. and Kuwata, S. Lipase-catalyzed transesterification procedure for the resolution of non-protein amino acids. Biotechnology Letters 14 (9) 789-794, 1992. PA1 5. Murata, M., Uchida, H. and Achiwa, K. Lipase-catalyzed enantioselective synthesis of optically active mephobarbital, hexobarbital and febarbamate. Chemical-Pharmaceutical Bulletin 40 (10) 2605-2609, 1992. PA1 6. Johnson, C. R., Golebiowski, A. and Steensma, D. H. Enzymatic asymmetrization in organic media--synthesis of unnatural glucose from cycloheptatriene. Journal of the American Chemical Society 114 (24) 9414-9418, 1992. PA1 7. Cruces, M. A., Otero, C., Bernabe, M., Martinlomas, M. and Ballesteros, A. Enzymatic preparation of acylated sucroses. Ann. New York Acad. Sci. (Enzyme Eng. XI, D. S. Clark, D. A. Estell, eds) 672 436-443, 1992. PA1 8. Tanaka, A., Fukui, T., Uejima, A., Zong, M. H. and Kawamoto, T. Bioconversion of nonnatural organic compounds--esterification and dehydrogenation of organosilicon compounds. Ann. New York Acad. Sci (Enzyme Eng. XI, D.S. Clark, D. A. Estell, eds) 672 431-435, 1992. PA1 9. Kodelia, G. and Kolisis, F. N. Studies on the reaction catalyzed by protease for the acylation of flavonoids in organic solvents. Ann. New York Acad. Sci. (Enzyme Eng. XI, D. S. Clark, D. A. Estell, eds) 672 451-457, 1992. PA1 10. Wagner, F., Kleppe, F., Lokotsch, W., Ziemann, A. and Lang, S. Synthesis of uncommon wax esters with immobilized lipases. Ann. New York Acad. Sci. (Enzyme Eng. XI, D. S. Clark, D. A. Estell, eds) 672 484-491, 1992. PA1 11. Patel. R. N., Howell, J. M., Banerjee, A., Fortney, K. F. and Szarka, L. J. Stereoselective enzymatic esterification of 3-benzoylthio-2-methylpropanoic acid. Ann. New York Acad. Sci (Enzyme Eng. XI, D. S. Clark, D. A. Estell, eds) 672 415-424, 1992. PA1 12. Bergbreiter, D. E. and Momongan, M. Asymmetric synthesis of organometallic reagents using enzymatic methods. Applied Biochemistry and Biotechnology 32 1-3 55-72, 1992. PA1 13. Carretero, J. C. and Dominguez, E. Lipase-catalyzed kinetic resolution of--hydroxy phenyl sulfones. Journal of Organic Chemistry 57 (14) 3867-3873, 1992. PA1 14. Johnson, C. R., Adams, J. P., Bis, S. J., Dejong, R. L., Golebiowski, A., Medich, J. R., Penning, T. D., Senanayake, C. H., Steensma, D. H. and Vanzandt, M. C. Applications of enzymes in the synthesis of bioactive polyols. Indian journal of Chemistry Section B--Organic Chemistry Including Medicinal Chemistry 32 (1) 140-144, 1993. PA1 15. Fabre, J., Betbeder, D., Paul, F., Monsan, P. and Perie, J. Regiospecific enzymic acylation of butyl a-D-glucopyranoside. Carbohydrate Research 243 (2) 407-411, 1993. PA1 16. Bianchi, D., Cesti, P., Golini, P., Spezia, S., Garavaglia, C. and Mirenna, L. Enzymatic preparation of optically active fungicide intermediates in aqueous and in organic media. Indian Journal of Chemistry Section B--Organic Chemistry Including Medicinal Chemistry 32 (1) 176-180, 1993. PA1 17. Kanerva, L. T. and Sundholm, O. Lipase catalysis in the resolution of racemic intermediates of diltiazem synthesis in organic solvents. Journal of the Chemical Society--Perkin Transactions I 13 1385-1389, 1993. PA1 18. Ikeda, I. and Klibanov, A. M. Lipase-catalyzed acylation of sugars solubilized in hydrophobic solvents by complexation. Biotechnology and Bioengineering 42 (6) 788-791, 1993. PA1 19. Hyun, C. K., Kim, J. H. and Ryu, D. D. Y. Enhancement effect of water activity on enzymatic synthesis of cephalexin. Biotechnology and Bioengineering 42 (7) 800-806, 1993. PA1 20. Panza, L., Luisetti, M., Crociati, E. and Riva, S. Selective acylation of 4,6-O-benzylidene glycopyranosides by enzymatic catalysis. J. Carbohydrate Chem. 12 (1) 125-130, 1993. PA1 21. Wang, L., Kobatake, E., Ikariama, Y. and Aizawa, M. Regioselective oxidative polymerization of 1,5-dihydroxynaphthalene catalyzed by bilirubin oxidase in a water-organic solvent mixed solution. Journal of Polymer Science Part A--Polymer Chemistry 31 (11) 2855-2861, 1993. PA1 22. Knani, D. and Kohn, D. H. Enzymatic polyesterification in organic media. 2. Enzyme-catalyzed synthesis of lateral-substituted aliphatic polyesters and copolyesters. J. Polymer Sci., Part A--Polymer Chem. 31 (12) 2887-2897, 1993. PA1 23. Kanerva, L. T. and Sundholm O. Enzymatic acylation in the resolution of methyl threo-2-hydroxy-3-(4-methoxyphenyl)-3-(2-x-phenylthio) propionates in organic solvents. Journal of the Chemical Society--Perkin Transactions I 20 2407-2410, 1993. PA1 24. Sharma, A. and Chattopadhyay, S. Lipase catalyzed acetylation of carbohydrates. Biotechnology Letters 15 (11) 1145-1146, 1993. PA1 25. Pavel, K. and Ritter, H. Enzymes in polymer chemistry. 7. Lipase-catalyzed esterification of carboxyl-terminated methacrylic oligomers and copolymers with isopropyl alcohol and 9-fluorenylmethanol. Makromolekulare Chemie--Macromolecular Chemistry and Physics 194 (12) 3369-3376, 1993. PA1 26. Uejima, A., Fukui, T., Fukusaki, E., Omata, T., Kawamoto, T., Sonomoto, K. and Tanaka, A. Efficient kinetic resolution of organosilicon compounds by stereoselective esterification with hydrolases in organic solvent. Appl. Microbiol. Biotech. 38 (4) 482-486, 1993. PA1 27. Mukesh, D., Sheth, D., Mokashi, A., Wagh, J., Tilak, J. M., Banerji, A. A. and Thakkar, K. R. Lipase catalyzed esterification of isosorbide and sorbitol. Biotechnology Letters 15 (12) 1243-1246, 1993. PA1 28. Baldessari, A., Iglesias, L. E. and Gros, E. G. Regioselective acylation of 3-mercaptopropane-1,2-diol by lipase-catalyzed transesterification. Journal of Chemical Research--S 9 382-383, 1993. PA1 29. De Goede, A. T. J. W., Benckhuijsen, W., van Rantwijk, F., Maat, L. and van Bekkum, H. Selective lipase-catalyzed 6-O-acylation of alkyl a-D-glucopyranosides using functionalized ethyl esters. Recueil Des Travaux Chimiques Des Pays Bas--Journal of the Royal Netherlands Chemical Society 112 (11) 567-572, 1993. PA1 30. Menendez, E. and Gotor, V. Acylation and alkoxycarbonylation of oximes through an enzymatic oximolysis reaction. Synthesis--Stuttgart 1 72-74, 1993. PA1 31. Li, Y. F. and Hammerschmidt, F. Enzymes in organic chemistry. 1. Enantioselective hydrolysis of a-(acyloxy) phosphonates by esterolytic enzymes. Tetrahedron--Asymmetry 4 (1) 109-120, 1993. PA1 32. Schlotterbeck, A., Lang, S., Wray, V. and Wagner, F. Lipase-catalyzed monoacylation of fructose. Biotechnology Letters 15 (1) 61-64, 1993. PA1 33. Frykman, H., Ohrner, N., Norin, T. and Hult, K. S-Ethyl thiooctanoate as acyl donor in lipase catalyzed resolution of secondary alcohols. Tetrahedron Letters 34 (8) 1367-1370, 1993. PA1 34. Oguntimein, G. B., Erdmann, H. and Schmid, R. D. Lipase catalyzed synthesis of sugar ester in organic solvents. Biotechnology Letters 15 (2) 175-180, 1993. PA1 35. Lopez, R., Perez, C., Fernandez-Mayorales, A. and Conde, S. Enzymatic transesterification of alkyl 2,3,4-tri-O-acyl-beta-D-xylopyranosides. J. Carbohydrate Chem. 12 (2) 165-171, 1993. PA1 36. Naemura, K., Ida. H. and Fukuda, R. Lipase YS-catalyzed enantioselective transesterification of alcohols of bicarbocyclic compounds. Bull. Chem. Soc. Japan 66 (2) 573-577, 1993. PA1 37. Lambusta, D., Nicolosi, G., Piattelli, M. and Sanfilippo, C. Lipase catalyzed acylation of phenols in organic solvents. Ind. J. Chem. Section B 32 (1) 58-60, 1993. PA1 38. Astorga, C., Rebolledo, F. and Gotor, V. Enzymatic hydrazinolysis of diesters and synthesis of N-aminosuccinimide derivatives. Synthesis--Stuttgart 3 287-289, 1993. PA1 39. Fukui, T., Kawamoto, T. and Tanaka, A. Enzymatic preparation of optically active silylmethanol derivatives having a stereogenic silicon atom by hydrolase-catalyzed enantioselective esterification. Tetrahedron--Asymmetry 5 (1) 73-82, 1994. PA1 40. Vazquez-Duhalt, R., Westlake, D. W. S. and Fedorak, P. M. Lignin peroxidase oxidation of aromatic compounds in systems containing organic solvents. Applied and Environmental Microbiology 60 (2) 459-466, 1994. PA1 41. Kodelia, G., Athanasiou, K. and Kolisis, F. N. Enzymatic synthesis of butyryl-rutin ester in organic solvents and its cytogenetic effects in mammalian cells in culture. Appl. Biochem. Biotech. 44 (3) 205-212, 1994. PA1 42. Tsai, S. W. and Wei, H. J. Effect of solvent on enantioselective esterification of naproxen by lipase with trimethylsilyl methanol. Biotechnology and Bioengineering 43 (1) 64-68, 1994. PA1 43. Athawale, V. D. and Gaonkar, S. R. Enzymatic synthesis of polyesters by lipase catalyzed polytransesterification. Biotechnology Letters 16 (2) 149-154, 1994. PA1 44. Janssen, G. G. and Haas, M. J. Lipase-catalyzed synthesis of oleic acid esters of polyethylene glycol 400. Biotechnology Letters 16 (2) 163-168, 1994. PA1 45. Kitazuma, T., Ikeya, T. and Murata, K. Synthesis of optically active trifluorinated compounds: asymmetric Michael additional with hydrolytic enzymes. Journal of Chemical Society, Chemical Communications 1331, 1986. PA1 1. Holland, H. L. Fungi as reagents in organic synthesis: preparation of some metabolites of prostanoids, steroids, and other natural products. Rev. Latinoam. Quim., (1st Spec. Suppl.), 318-329, 1990. PA1 2. Sih, C. J. and Rosazza, J. P. Microbial transformations in organic synthesis. Tech. Chem. (N.Y.), 10 (Appl. Biochem. Syst. Org. Chem., Part 1) 69-106, 1976. PA1 3. Sih, C. J. and Chen, C. S. Microbial asymmetric catalysis-enantioselective reduction of ketones. Angew. Chem. Int. Ed. Enal. 23 (8) 570-578, 1984. PA1 4. Thompson, L. A. and Knowles, C. J., Linton, E. A. and Wyatt, J. M. Microbial biotransformations of nitrites. Chem. Br. 24 (9) 900, 1988. PA1 5. Servi, S. Baker's yeast as a reagent in organic synthesis. Synthesis 1 1-25, 1990. PA1 6. Csuk, R. and Glanzer, B. I. Baker's yeast mediated transformations in organic chemistry. Chem. Rev. 91 (1) 49-97, 1991. PA1 7. Roberts, S. M., Wiggins, K. and Casy, G. Whole cells and isolated enzymes in organic synthesis. Wiley, New York, 1992. PA1 8. Ward, O. P. and Young, C. S. Reductive biotransformations of organic compounds by cells or enzymes of yeast. Enzyme Microb. Technol. 12 (7) 482-493, 1990. PA1 1. Jones, J. B. Enzymes in organic synthesis. Tetrahedron 42 (13) 3351-3405, 1986. PA1 2. Yamada, H. and Shimizu, S. Microbial and enzymatic processes for the production of biologically and chemically useful compounds. Angew. Chem. Int. Ed. Engl. 27 (5) 622-642, 1988. PA1 3. Roberts, S. M. Enzymes as catalysts in organic synthesis. NATO ASI Ser., Ser. A. 178 443-463, 1989. PA1 4. Chen, S. S. and Sih, C. J. General aspects and optimization of enantioselective biocatalysis in organic solvents: the use of lipases. Angew. Chem (Int. Ed. Engl. 28, n 6 695-707) 101 (6) 711-724, 1989. PA1 n is an integer of 2-10; and PA1 R.sub.2 is hydrogen, C.sub.1-10 alkyl, C.sub.1-10 alkenyl, or a 6 substituted saccharide compound selected from the group consisting of glucose, galactose, allose, altrose, mannose, gulose, idose, talose, lactose, cellobiose, sucrose, fructose and maltose, in the open or pyranose form. PA1 where n is an integer of 2-10; and PA1 R.sub.10 is OH, C.sub.1-10 alkoxy, C.sub.1-10 alkenyloxy, a 6 substituted saccharide compound selected from the group consisting of glucose, galactose, allose, altrose, mannose, gulose, idose, talose, lactose, cellobiose, sucrose, fructose and maltose, in the open or pyranose form, NR.sub.11 R.sub.12 where R.sub.11 and R.sub.12 are each independently hydrogen, C.sub.1-10 alkyl, substitued C.sub.1-10 alkyl substitued with C.sub.1-10 alkyl, C.sub.6-20 aryl or halogen; PA1 R.sub.7 is H, OH, F, Cl, Br, or I; PA1 R.sub.8 and R.sub.9 are each independently hydrogen or C.sub.1-10 alkyl, PA1 provided that when R.sub.7 is H, R.sub.4, R.sub.5 and R.sub.6 are not each hydrogen. PA1 R.sub.14 is CH.sub.3 or CO.sub.2 H; and PA1 R.sub.15 is hydrogen or CO(CH.sub.2).sub.n COR.sub.16 PA1 where n is an integer of 2-10; and PA1 R.sub.16 is OH, C.sub.1-10 alkoxy, C.sub.1-10 alkenyloxy, a 6 substituted saccharide compound selected from the group consisting of glucose, galactose, allose, altrose, mannose, gulose, idose, talose, lactose, cellobiose, sucrose, fructose and maltose, NR.sub.17 R.sub.18 where R.sub.17 and R.sub.18 are each independently hydrogen, C.sub.1-10 alkyl, substitued C.sub.1-10 alkyl substitued with C.sub.1-10 alkyl, C.sub.6-20 aryl or halogen; PA1 provided that when R.sub.13 and R.sub.15 are each hydrogen, R.sub.14 is not CH.sub.3.
References Demonstrating that Enzyme Enantioselectivity can be Changed/Tailored
References Demonstrating the Ability of Enzymes to Convert Unnatural Substrates
References Demonstrating Microbial Transformations
Reviews
Taxol.sup.1, a diterpenold originally isolated from the bark of the Pacific yew, Taxus brevifolia, is a powerful antimitotic agent.sup.2 that acts by promoting tubulin assembly into stable aggregated structures. Taxol has shown tremendous potential as an anticancer compound. Indeed, it is now used for the treatment of refractory ovarian cancer.sup.3, and clinical trials are encouraging for the treatment of breast, lung.sup.4, head, and necks.sup.6 cancers. Because of its broad antitumor activity and limited availability, numerous studies have been devoted to the synthesis.sup.6 (including semisynthesis from the baccatin III nucleus.sup.7), mechanism.sup.1, and structure-activity relationships of taxol and protaxols..sup.1a,1d,8 Despite such intense investigation, the use of taxol as an anticancer drug is compromised by its poor aqueous solubility. For this reason, a number of water-soluble taxol prodrugs have been synthesized that contain hydrophilic or charged functionalities attached to specific sites on the taxol molecule ..sup.9
Acylation at the 2' position can be a very effective strategy for improving the water solubility of taxol..sup.9a-e Interestingly, acylation of the C-2' hydroxyl eliminates microtube stabilization but not cytotoxicity, which is consistent with the hydrolytic regeneration of taxol from protaxol within the cell..sup.8 Water soluble protaxols modified at the 2' position include arylsulfonyl ethoxycarbonates and thiodiglycolic esters synthesized by Nicolaou et al..sup.9a, the most soluble of which were 100 to 1000 times more soluble than taxol.
Accordingly, taxol derivatives with improved solubiity are sought.